Lipopolysaccharide (LPS)-responsive beige-like anchor (LRBA) was initially identified as an LPS-upregulated gene, encoding a 2863-amino acid, multiple-protein, as seen in FIG. 1 (Kerr, W. G., Heller, M. & Herzenberg, L. A. Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains. Proc Natl Acad Sci USA 93, 3947-52 (1996); Wang, J. W., Howson, J., Haller, E. & Kerr, W. G. Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. J Immunol 166, 4586-95 (2001)). Human LRBA and murine Lrba proteins are 90% identical (2587/2859) with 94% positive (2690/2859) amino acid homology. Three murine Lrba isoforms with differences at the C-terminal were identified (Wang, et al. Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. 2001. J Immunol 166, 4586-95), while the human LRBA has two major isoforms (Wang, et al., Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. 2001. J Immunol 166, 4586-95; Lopez-Herrera, et al., Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity. 2012. Am J Hum Genet 90, 986-1001).
LRBA is mainly localized to Golgi complex (GC), but also found in cell membrane, endosomes and vesicles. This is similar to neurobeachin (Nbea) (Wang, J. W. et al. Deregulated expression of LRBA facilitates cancer cell growth. Oncogene 23, 4089-97 (2004); Wang, X. et al. Neurobeachin: A protein kinase A-anchoring, beige/Chediak-higashi protein homolog implicated in neuronal membrane traffic. 2000. J Neurosci 20, 8551-65), an autism candidate gene and isoform of LRBA, has very similar localizations to that of LRBA and is implicated in post-Golgi membrane traffic and regulatory secretion pathway of large dense-core vesicles containing growth factors and hormones (Volders, et al., Drosophila rugose is a functional homolog of mammalian Neurobeachin and affects synaptic architecture, brain morphology, and associative learning. 2012. J Neurosci 32, 15193-204; Castermans, et al., SCAMPS, NBEA and AMISYN: three candidate genes for autism involved in secretion of large dense-core vesicles. 2010. Hum Mol Genet 19, 1368-78), isoform, Neurobeachin (Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97; Wang, et al., Neurobeachin: A protein kinase A-anchoring, beige/Chediak-higashi protein homolog implicated in neuronal membrane traffic. 2000. J Neurosci 20, 8551-65).
The exact function of LRBA is unknown. LRBA has structural similarity to lysosomal trafficking regulator (LYST) and potentially is an A-kinase anchoring protein (AKAP)(1). It belongs to the WDL-BEACH-WD40 (WBW) gene family containing the WBW super domain. The WBW proteins appear to function as scaffolding proteins in vesicle trafficking (Wang, et al., Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. 2001. J Immunol 166, 4586-95; Lopez-Herrera, et al., Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity 2012. Am J Hum Genet 90, 986-1001; Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97) and are important in human diseases (Cullinane, A. R., Schaffer, A. A. & Huizing, M. The BEACH Is Hot: A LYST of Emerging Roles for BEACH-Domain Containing Proteins in Human Disease. 2013. Traffic 14(7):749-66). There are nine WBW human family members, which all appear to function as scaffolding proteins in vesicle trafficking regulation, involved in lysosome size regulation; LRBA, lysosomal trafficking regulator (LYST), neutral sphingomyelinase activation associated factor (NSMAF), WD and FYVE zinc finger domain containing protein 3 (WDFY3), WDFY4, neurobeachin-like 1 (NBEAL1), neurobeachin-like 2 (NBEAL2), neurobeachin (NBEA), WD repeat domain 81 (WDR81). They appear to be involved in vesicle trafficking as scaffolding proteins and regulating lysosome size (LYST and NSMAF), apoptosis (NSMAF, LRBA), autophagy (LYST, WD and WDFY3, LRBA), granule size (LYST, NBEAL2, and NBEA), or synapse formation (NBEA).
These family members are important in human diseases. For example, mutations of four WBW genes (LYST, LRBA, NBEAL2 and WDFY4) cause recessive Mendelian diseases and NBEA is a current candidate for autism (Cullinane, A. R., Schaffer, A. A. & Huizing, M. The BEACH Is Hot: A LYST of Emerging Roles for BEACH-Domain Containing Proteins in Human Disease. 2013. Traffic; 14(7):749-66)). The WD and FYVE zinc finger domain containing protein 4 (WDFY4) is strongly associated with SLE in Asian populations. NSMAF is a TNF adaptor protein which is required for the TNF-induced expression of cytokines, such as IL-6 and CXCL-2 (Adam-Klages, et al., FAN, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. 1996. Cell 86, 937-47; Montfort, et al., FAN stimulates TNF(alpha)-induced gene expression, leukocyte recruitment, and humoral response. 2009. J Immunol 183, 5369-78).
Accordingly, LRBA is extensively associated with the endomembrane system and vesicle trafficking. Vesicle trafficking is essential for deposition and signal transduction of membrane proteins and protein secretions, which also controls proper functioning of the immune system.
NBEA, which has 75% of protein homology with LRBA, is an isoform of LRBA linked to autism, platelet development, obesity, and multiple myeloma in humans (O'Neal, et al., Neurobeachin (NBEA) is a target of recurrent interstitial deletions at 13q13 in patients with MGUS and multiple myeloma. 2009. Exp Hematol 37, 234-44) and body length, synaptic spine patterns of neurons, and obesity in mice (Cullinane, et al., The BEACH Is Hot: A LYST of Emerging Roles for BEACH-Domain Containing Proteins in Human Disease. 2013. Traffic; 14(7):749-66). It has very similar subcellular localizations to that of LRBA and is implicated in post-Golgi membrane traffic and the regulatory secretion pathway of large dense-core vesicles containing growth factors and hormones (Wang, J. W. et al. Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97; Volders, et al., Drosophila rugose is a functional homolog of mammalian Neurobeachin and affects synaptic architecture, brain morphology, and associative learning. 2012. J Neurosci 32, 15193-204; Castermans, et al., SCAMPS, NBEA and AMISYN: three candidate genes for autism involved in secretion of large dense-core vesicles. 2010. Hum Mol Genet 19, 1368-78; Wang, et al., Neurobeachin: A protein kinase A-anchoring, beige/Chediak-higashi protein homolog implicated in neuronal membrane traffic. 2000. J Neurosci 20, 8551-65). NBEA binds to an important signaling complex, protein kinase A, and acts as a negative regulator of vesicle secretion, as does LRBA ortholog Beige Protein Homolog 1 (Bph1) in yeast. NBEA homozygous knockout mice died perinatally due to the lack of synaptic transmissions (Su, et al., Neurobeachin is essential for neuromuscular synaptic transmission. 2004. J Neurosci 24, 3627-36; Medrihan, et al., Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses. 2009. Journal of Physiology-London 587, 5095-5106), suggesting the protein is important in trafficking cargo proteins to pre- and post-synaptic compartments (Niesmann, K. et al. Dendritic spine formation and synaptic function require neurobeachin. 2011. Nat Commun 2:557).
LRBA orthologues Bph1 and rugose (rg) (Drosophila) are not essential and is both cytosolic and membrane peripherally bound. Deletion of Bph1 increases secretion of carboxypeptidase Y in Saccharomyces cerevisiae and missorting of alkaline phosphatase. The vacuole morphology is not affected by disruption or overexpression of Bph1. The growth of the delta Bph1 strain is impaired by low pH, potassium acetate or calcofluor white, a fluorescent stain that binds strongly to the cell wall which contains cellulose and chitin, probably due to a defect on trafficking from the GC. Genetically, Bph1 interacts with VPS9, FLO1, FLO9, BTS1, OKP1, VPS9, BTS1 and OKP1. These data suggest that Bph1 is involved in protein sorting and cell wall formation (Shiflett, et al., Bph1p, the Saccharomyces cerevisiae homologue of CHS1/Beige, functions in cell wall formation and protein sorting. 2004. Traffic 5, 700-710). Mutations of rg in Drosophila are embryonic semi-lethal, exhibiting a reduced lifespan of the organism and severe rough eye phenotype, caused by cell type-specific apoptosis with increased Jun N-terminal kinase activity and decreased EGFR signaling activity (Wech & Nagel, Mutations in rugose promote cell type-specific apoptosis in the Drosophila eye. 2005. Cell Death Differ 12, 145-52). Genetically, rg interacts with 14 genes, including multiple components of EGFR, Notch, RAS and MAPK pathways (Schreiber, et al., Genetic screen for modifiers of the rough eye phenotype resulting from overexpression of the Notch antagonist hairless in Drosophila. 2002. Genesis 33, 141-52; Shamloula, et al., rugose (rg), a Drosophila A kinase Anchor Protein, Is Required for Retinal Pattern Formation and Interacts Genetically With Multiple Signaling Pathways. 2002. Genetics 161, 693-710). Similarly, LRBA appears to interact with multiple important signal transduction pathways, including EGFR, Notch, PKA, Ras, E2F1, p53, MAPK, NF-κB, cytokines, immunoglobulins (IgG, IgA, IgE and IgM etc.), CVID receptors and TLRs (Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97; de Souza, et al., SEL-2, the C. elegans neurobeachin/LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells. 2007. Development 134, 691-702; Schreiber, et al., Genetic screen for modifiers of the rough eye phenotype resulting from overexpression of the Notch antagonist hairless in Drosophila. 2002. Genesis 33, 141-52; Shamloula, et al., rugose (rg), a Drosophila A kinase Anchor Protein, Is Required for Retinal Pattern Formation and Interacts Genetically With Multiple Signaling Pathways. 2002. Genetics 161, 693-710).
LRBA orthologue in Dictyostelium is essential for cytokinesis (Kwak, et al., LvsA, a protein related to the mouse beige protein, is required for cytokinesis in Dictyostelium. 1999. Mol Biol Cell 10, 4429-39). LRBA orthologue, sel-2, also called F10F2.1, in Caenorhabditis elegans, is a negative regulator of lin-12/Notch activity in the vulval precursor cells, which are polarized epithelial cells maintained through regulated activity of the basolateral LET-23/EGF receptor and apical LIN-12/Notch. Loss of sel-2 activity causes basolateral mislocalization and increased accumulation of LIN-12/Notch and basolateral LET-23/EGF, indicating that SEL-2 is involved in endosomal traffic and may be involved in the efficient delivery of cell surface proteins to the lysosome (de Souza, et al., SEL-2, the C. elegans neurobeachin/LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells. 2007. Development 134, 691-702). Two RII binding motifs were predicted for sel-2, both human and murine LRBA, by aligning with the known B1 and B2 PKA RII tethering sites in rg2.
Mutations of LRBA cause common variable immunodeficiency and autoimmunity, resulting in LRBA's synonym name, common variable immune deficiency-8 (CVID8). Six germ lines of LRBA mutations reported to date cause common variable immunodeficiency (CVID) with autoimmunity, manifested as hypogammaglobulinemia, antibody deficiency, defective B-cell differentiation, recurrent bacterial infections, particularly respiratory infections, and variable autoimmune disorders including idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, and inflammatory bowel disease (Spickett, et al., Common variable immunodeficiency: how many diseases? 1997. Immunol Today 18, 325-8). Other CVID gene mutations include members of the B cell coreceptor complex (CD1919, CD2120 and CD8121), CD2022, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI; Salzer, et al. Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans. 2005. Nat Genet 37, 820-8) and B cell-activating factor receptor (BAFFR; Warnatz, K. et al. B-cell activating factor receptor deficiency is associated with an adult-onset antibody deficiency syndrome in humans 2009. Proc Natl Acad Sci USA 106, 13945-50), and inducible costimulator (ICOS; Grimbacher, et al. Homozygous loss of ICOS is associated with adult-onset common variable immunodeficiency. 2003. Nat Immunol 4, 261-8). The discoveries of CVID-causing mutations of these genes show that a monogenic defect may produce the whole spectrum of CVID, and that it is possible to unravel the genetic causes underlying most human diseases thought to be polygenic (Yong, et al., The role of costimulation in antibody deficiencies: ICOS and common variable immunodeficiency. 2009. Immunol Rev 229, 101-13).
It is caused by defective B cell differentiation and impaired secretion of immunoglobulins (Saiki, et al., Three distinct stages of B-cell defects in common varied immunodeficiency. 1982. Proc Natl Acad Sci USA 79, 6008-12; Bryant, et al., Classification of patients with common variable immunodeficiency by B cell secretion of IgM and IgG in response to anti-IgM and interleukin-2. 1990. Clin Immunol Immunopathol 56, 239-48). CVID is a diagnosis of exclusion and is highly heterogeneous, genetically, immunologically and clinically (Gathmann, et al., The European internet-based patient and research database for primary immunodeficiencies: update 2011. 2012. Clin Exp Immunol 167, 479-91; Eibel, et al., Common variable immunodeficiency at the end of a prospering decade: towards novel gene defects and beyond. 2010. Curr Opin Allergy Clin Immunol 10, 526-33; Park, et al., Common variable immunodeficiency: a new look at an old disease. 2008. Lancet 372, 489-502). About two-third of CVID subjects have an autoimmune problem17, most commonly autoimmune hemolytic anemia (AHA), autoimmune thrombocytopenia, rheumatoid arthritis, and pernicious anemia (Cunningham-Rundles& Bodian, Common variable immunodeficiency: clinical and immunological features of 248 patients. 1999. Clin Immunol 92, 34-48). The etiology of about 80% of CVID remains unknown18, although over the past ten years, significant progress has been made in elucidating genetic mechanisms that result in a CVID phenotype.
Unlike the other seven CVID proteins, which are usually small (˜27 kD) and localized on the cell membrane, LRBA is a 319 kD multiple-domain protein (Kerr, et al., Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains. 1996. Proc Natl Acad Sci USA 93, 3947-52; Wang, et al., Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. 2001. J Immunol 166, 4586-95) and A kinase anchor protein (AKAP), which could serve as a scaffold to interact with multiple proteins. Additionally, most CVID genes are B cell membrane receptors, except for ICOS which is on T cells, while LRBA is ubiquitously expressed as a vesicle trafficking regulator, required for homeostasis and activation of plasma membrane receptors (Wang, et al., Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. 2001. J Immunol 166, 4586-95; Cullinane, et al., The BEACH Is Hot: A LYST of Emerging Roles for BEACH-Domain Containing Proteins in Human Disease. 2013. Traffic; 14(7):749-66). Thus, LRBA may regulate other CVID genes, for example, CD19, CD20 and BAFFR, because their levels are low when LRBA is absent (Lopez-Herrera, et al., Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity. 2012. Am J Hum Genet 90, 986-1001). Further, LRBA deficiency causes both immunodeficiency and autoimmunity (Lopez-Herrera, et al., Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity 2012. Am J Hum Genet 90, 986-1001; Burns, et al., LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia. 2012. J Allergy Clin Immunol 130, 1428-32; Parvaneh, et al., Primary immunodeficiencies: a rapidly evolving story. 2013. J Allergy Clin Immunol 131, 314-23). All 11 LRBA deficient CVID subjects identified thus far have autoimmune diseases, as seen in Table 1. TACI mutations also are associated with autoimmunity but to a lesser degree (36% vs. 23% of patients with wild-type TACI) (Salzer, et al., Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes. 2009. Blood 113, 1967-76). LRBA is the only CVID protein that is a protein kinase A anchor and thus can function as protein kinase A (PKA) to regulate protein activity by phosphorylation. In addition to these above unique features, LRBA also is unique in regulating autophagy, apoptosis, membrane dynamics and receptor signaling, all of which are important for inflammation (Kerr, et al., Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains. 1996. Proc Natl Acad Sci USA 93, 3947-52; Wang, et al., Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. 2001. J Immunol 166, 4586-95). The average age of symptom onset is three years, earlier than the mean age of 26.3 years for most CVID. In addition to immunological disorders, LRBA-deficient patients have an array of other medical problems which include: retarded growth, failure to thrive, growth hormone deficiency, asthma, monoarthritis, seizure disorders, granulomatous infiltration, finger clubbing, hepatosplenomegaly, allergic dermatitis, and nephrotic syndrome (Lopez-Herrera, et al., Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity 2012. Am J Hum Genet 90, 986-1001; Burns, et al., LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia. 2012. J Allergy Clin Immunol 130, 1428-32; Alangari, et al., LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency. 2012. J Allergy Clin Immunol 130, 481-8 e2; Cunningham-Rundles & Bodian, Common variable immunodeficiency: clinical and immunological features of 248 patients. 1999. Clin Immunol 92, 34-48).
TABLE 1Clinical features of LRBA-deficient patientsClinical conditionsManifestationsPatient 1-5 from the study1Patient 1AutoimmuneIdiopathic thrombocytopenic purpura (ITP).TreatmentIntravenous immunoglobulin (IVIG) replacementImmunologiclow immunoglobulin levelsinvestigationsNon-immunologicalSeverely retarded growth, significant clubbing, strabismus as adisordersresult of abducens nerve palsy along with hemiplegia, cerebralmass.Recurrent infectionsLymphoid interstitial pneumonia (LIP). Pleuropneumonia,chronic lung disease, bilateral bronchiectasis.Patient 2AutoimmuneSelf-limiting ITP; Asthma satisfactorily treated with inhaledsteroids. Reactive monoarthritis responded well to local steroidinjections.ImmunologicLow immunoglobulin levels.investigationsNon-immunologicalGrowth retarded (both weight and height were below the fifthdisorderspercentile since the age of 2).Recurrent infectionsSerous otitis media, massive pneumonia along with loculatedempyema, chronic lung disease with bilateral bronchiectasis.Patient 3Chronic diarrheaSevere diarrhea with no detectable bacterial or parasitic infection.AutoimmuneITP, lymphadenopathy, autoimmune haemolytic anemia (AIHA),atrophic gastritis with autoantibodies against intrinsic factor, anda submaxillar abscess, granulomatous infiltration with T cells,plasma cells, and macrophages but showed low B cell numbers.ImmunologicA moderate IgG hypogammaglobulinemia and complete IgAinvestigationsdeficiency but a normal neutrophil count; intermittently elevatedIgM; swelling of hilar and mediastinal lymph nodes with a mixedlymphoid follicular hyperplasia with the absence of the follicularmantle zone.Recurrent infectionsPerineal molluscum contagiosum, recurrent warts, recurrent mildrespiratory infections, severe recurrent pneumonias, includingseveral interstitial pneumonias, a lymphoid interstitial pneumoniaand bronchiectasis.TreatmentThe interstitial lung disease was treated with methylprednisolone.Attempts to taper steroids were frequently associated withrelapses, and long-term treatment with infliximab was initiatedand allowed the discontinuation of methylprednisolone.Infliximab had little improvement of the chronic diarrhea. IVIGreplacement.Patient 4Chronic diarrheaITP, AIHA and autoimmune enteropathy (which can be classifiedand autoimmune as Crohn disease)Non-immunologicalFailure to thrive.disordersImmunologicLow immunoglobulin levels.investigationsTreatmentIVIG replacement and antibiotic prophylaxisRecurrent infections Recurrent upper-respiratory-tract infections, several episodes ofpneumonia recurrent respiratory and gastrointestinal infections,severe lower respiratory-tract infections along with fingerclubbing, hepatosplenomegaly an obstruction of the small airwaysand bronchiectasis, recurrent conjunctivitis and urticaria and acorpulmonale with consecutive right-heart failure.TreatmentAntibacterials, antifungals, and IVIG.Patient 5Chronic diarrheaRecurrent chronic diarrhea, autoimmune phenomena,and autoimmunehypothyroidism, and or AIHA allergic dermatitis, intestinalinflammation and subtotal villous atrophy, autoimmunephenomena, hypothyroidism, and myasthenia gravis. no signs ofITP or AIHA, bronchiectasis; Died at the age of 19 afterrespiratory failureNon-immunologicalRetarded growthdisordersImmunologicSelective IgA and IgG2 deficiency, IgG and IgM levels declinedinvestigationsgradually.Recurrent infectionsRecurrent upper-respiratory-tract infections, including sinusitisand otitis media, pneumonia.Patient 6-10 from the study2Patient 6ImmunologicNormal serum IgG, IgA, IgM, and IgE levels and normal numbersinvestigationsof CD3+ T lymphocytes, CD4+ and CD8+ T-cell subsets, B cells,and natural killer (NK) cells. Normal T-cell proliferation to themitogens PHA and ConA and a normal increase in antibody titersafter vaccination with tetanus toxoid, diphtheria, toxoid andHaemophilus influenzae capsular antigens.Recurrent infectionsno history of recurrent infectionsTreatmentResponded to intravenous immunoglobulin (IVIG) replacementtherapy and a short course of prednisone.Patient 7Chronic diarrheaNephrotic syndrome; mucous non-bloody stools; lymphocyticand autoimmuneinfiltration of the lamina propria; villous atrophy and markedinflammation.TreatmentPrednisone and azathioprine with poor compliance; monthlyintramuscular vitamin B12 injections; human growth hormonereplacement.ImmunologicNormal serum IgG, IgA, IgM, and IgE levels and normal numbersinvestigationsof CD3+ T lymphocytes, CD4+ and CD8+ T-cell subsets, Blymphocytes, and NK cells, normal T-cell proliferation to themitogens PHA and ConA and a normal increase in antibody titersafter vaccination with pneumococcal vaccine.Non-immunologicalClubbing; growth hormone deficiency.disordersRecurrent infectionsNo history of recurrent infections.AnemiaMegaloblastic anemia.Patient 8Chronic diarrheaNon-mucous and non-bloody chronic diarrhea; mucosaland autoimmuneinflammation with lymphocytic infiltration but no granulomas orulcerations; recurrent arthritis in the large joints, mainly the kneeswith inflammation.TreatmentChronic diarrhea improved on oral prednisone.ImmunologicLow serum IgG and IgA levels and decreased B-cell numbers butinvestigationsnormal numbers of T cells, T-cell subsets, and NK cells; markedlyreduced T-cell proliferation in response to PHA and anti-CD3mAb.Recurrent infectionsRecurrent otitis media and pneumonia; bilateral bronchiectasisand finger clubbing.Patient 9Chronic diarrheaChronic non-bloody and non-mucous diarrhea; duodenal villousand autoimmuneatrophy; autoimmune thrombocytopenia and autoimmunehemolytic anemia both of which responded to treatment withsteroids and rituximab.ImmunologicLow serum IgG and IgA levels and decreased B-cell numbers butinvestigationsnormal numbers of T cells, T-cell subsets, and NK cells; markedlyreduced T-cell proliferation in response to PHA and anti-CD3mAb.Recurrent infectionsno history of recurrent infectionsPatient 10Chronic diarrheaChronic diarrhea with no blood or mucus.ImmunologicLow serum IgG and IgA levels and normal numbers of T cells, investigationsT-cell subsets, B cells, and NK cells; markedly reduced T-cellproliferation in response to PHA and anti-CD3 mAb.Recurrent infectionsno history of recurrent infections or autoimmune hematologicmanifestations.Patient 11 thestudy 8Chronic diarrheaChronic diarrhea associated with an autoimmune enteropathy,and autoimmuneduodenal villous atrophy and large bowel lymphocyticinfiltration; erythema nodosum, transient arthritis of both feet, andrecurrent hemolytic anemia; extensive lung infiltration of amixture of CD3+ T and CD20+ B cells.ImmunologicRaised IgG levels, raised inflammatory markers, and a lowinvestigationsnumber of natural killer cells; normal lymphocyte subsets,double-negative T cells, T-cell proliferation assays, IgA, IgM,tetanus vaccine responses, and a nitroblue tetrazolium test;lymphadenopathy, splenomegaly, neutropenia, andthrombocytopenia, antineutrophil antibodies; normal CD 19+ Bcells and IgG level, a new-onset antibody deficiency with absentvaccine responses.TreatmentSeveral courses of steroids, rituximab (with prophylacticimmunoglobulin replacement), and mycophenolate mofetil.Non-immunologicalGrowth failure.disordersRecurrent infectionsNo significant history of infections except for a psoas abscessassociated with chronic neutropenia; five years after initialpresentation, after multiple courses of rituximab, developedrecurrent infections, after withdrawal of immunoglobulin therapy.1Lopez-Herrera, et al., Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity. 2012. Am J Hum Genet 90, 986-1001.2Alangari, et al., LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency. 2012. J Allergy Clin Immunol 130, 481-8 e2.
As LRBA is structurally conserved throughout the eukaryotic kingdom, it may be involved in pathways similar to its orthologues and paralogues. Mutations in lysosomal trafficking regulator (LYST), which is a paralogue gene of LRBA, causes Chediak-Higashi syndrome (CHS), and A-kinase anchoring proteins (Kerr, et al., Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains. 1996. Proc Natl Acad Sci USA 93, 3947-52; Wang, et al., Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. 2001. J Immunol 166, 4586-95). CHS is characterized by severe immunodeficiency and premature mortality (Faigle, et al., Deficient peptide loading and MHC class II endosomal sorting in a human genetic immunodeficiency disease: the Chediak-Higashi syndrome. 1998. J Cell Biol 141, 1121-34; Barrat, et al., Defective CTLA-4 cycling pathway in Chediak-Higashi syndrome: a possible mechanism for deregulation of T lymphocyte activation. 1999. Proc Natl Acad Sci USA 96, 8645-50). LRBA and LYST may have similar functions, as they have high similarity of protein structure. Three independent papers show that absence of LRBA, resulting from homozygous mutations or deletions, causes autosomal recessive diseases of severe immunodeficiency (defective B-cell differentiation, hypogammaglobulinemia, recurrent infections), autoimmunity (idiopathic thrombocytopenic purpura (ITP), autoimmune hemolytic anemia and inflammatory bowel disease) (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity 2012. Am J Hum Genet 90, 986-1001; Burns, et al., LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia. 2012. J Allergy Clin Immunol 130, 1428-32; Alangari, et al., LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency. 2012. J Allergy Clin Immunol 130, 481-8 e2), and inflammatory cell infiltration (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity 2012. Am J Hum Genet 90, 986-1001; Burns, et al., LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia. 2012. J Allergy Clin Immunol 130, 1428-32; Alangari, et al., LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency. 2012. J Allergy Clin Immunol 130, 481-8 e2; Faigle, et al., Deficient peptide loading and MHC class II endosomal sorting in a human genetic immunodeficiency disease: the Chediak-Higashi syndrome. 1998. J Cell Biol 141, 1121-34; Barrat, et al., Defective CTLA-4 cycling pathway in Chediak-Higashi syndrome: a possible mechanism for deregulation of T lymphocyte activation. 1999. Proc Natl Acad Sci USA 96, 8645-50; Alangari, et al., LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency. 2012. J Allergy Clin Immunol 130, 481-8 e2). Another analogue, rg interacts with 14 genes, including multiple components of EGFR, Notch, RAS and MAPK pathways (Shamloula, et al., rugose (rg), a Drosophila A kinase Anchor Protein, Is Required for Retinal Pattern Formation and Interacts Genetically With Multiple Signaling Pathways. 2002. Genetics 161, 693-710). LRBA interacts with the EGFR pathway (Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97).
The GC is now viewed as headquarters for signal transduction and cell-fate decisions, in addition to its “classic role” of cargo sorting/processing and metabolism (Wilson, C. et al. The Golgi apparatus: an organelle with multiple complex functions. Biochem J 433, 1-9; Wilson, C. et al. The Golgi apparatus: an organelle with multiple complex functions. Biochem J 433, 1-9). For example, internalized LPS and Toll-like receptor-4 (TLR-4) are found in GC (Hornef, M. W., Frisan, T., Vandewalle, A., Normark, S. & Richter-Dahlfors, A. Toll-like receptor 4 resides in the Golgi apparatus and colocalizes with internalized lipopolysaccharide in intestinal epithelial cells. J Exp Med 195, 559-70 (2002)). Activation of the proto-oncogene Ras on the Golgi can lead to cell transformation (Bivona, T. G. et al. Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature 424, 694-8 (2003); Tracey, K. J. et al. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330, 662-4 (1987)). The proto-oncogenes Ras and Src are activated on the GC (Pulvirenti, et al., A traffic-activated Golgi-based signalling circuit coordinates the secretory pathway. Nat Cell Biol 10, 912-22 (2008); Bivona, T. G. et al. Phospholipase Cgamma activates Ras on the Golgi apparatus by means of RasGRP1. Nature 424, 694-8 (2003); Pulvirenti, et al., A traffic-activated Golgi-based signalling circuit coordinates the secretory pathway. Nat Cell Biol 10, 912-22 (2008)). Membrane trafficking is crucial in transducing signals to GC and other cellular locations (Kholodenko, Four-dimensional organization of protein kinase signaling cascades: the roles of diffusion, endocytosis and molecular motors. J Exp Biol 206, 2073-82 (2003)), as simple diffusion has a limited role in intracellular transport of signaling complexes (Kholodenko, Four-dimensional organization of protein kinase signaling cascades: the roles of diffusion, endocytosis and molecular motors. J Exp Biol 206, 2073-82 (2003)). Thus the endomembrane system has critical role in signal transduction, which is not yet appreciated enough. LRBA may be important in GC's function, as LRBA is extensively co-localized with GC proteins and an abnormally high number of GCs is found in LRBA deficient B cells (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. Am J Hum Genet 90, 986-1001 (2012)). It is extensively associated with the endomembrane system and vesicle trafficking. Vesicle trafficking is essential for deposition, secretion and signal transduction of immune effectors.
Taken together, LRBA appears to be critical in vesicle trafficking involving deposition of membrane proteins, endocytosis of signal transduction complex and protein secretion, and thus a crucial regulator of these proteins including the other CVID proteins, TLRs and antibodies, involved in multiple pathways and cellular processes such as growth, apoptosis, autophagy, membrane dynamics, receptor signaling, and cell differentiation (Wang, et al., Identification of a novel lipopolysaccharide-inducible gene with key features of both A kinase anchor proteins and chs1/beige proteins. 2001. J Immunol 166, 4586-95). Deficiency of LRBA may cause vesicle trafficking defects and affect many membrane or secreted immune effectors, resulting in broader and severer problems, even death (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. 2012. Am J Hum Genet 90, 986-1001; Burns, et al., LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia. 2012. J Allergy Clin Immunol 130, 1428-32; Alangari, et al., LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency. 2012. J Allergy Clin Immunol 130, 481-8 e2).
A well characterized LRBA polyclonal antibody and organelle-specific antibodies were used in the present study to detect endogenous proteins and study their co-localizations. The results show that LRBA is extensively associated with the endomembrane system, including the Golgi complex, endosomes, lysosomes, plasma membranes, nucleus, pseudopodia and microtubules, and vesicle trafficking, which is responsive to LPS stimulation. These results suggest that LRBA plays a role in vesicle trafficking and signal transduction essential for the immune system (Burns, et al., LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia. 2012. J Allergy Clin Immunol 130, 1428-32).
It is proposed that LRBA functions as a converging node for multiple pathways through vesicle trafficking and plays a fundamental role in the immune system. This concept is supported by data showing LRBA may interact with multiple important signal transduction pathways (Shamloula, et al. rugose (rg), a Drosophila A kinase Anchor Protein, Is Required for Retinal Pattern Formation and Interacts Genetically With Multiple Signaling Pathways. 2002. Genetics 161, 693-710); mutations of LRBA cause severe immunodeficiency and autoimmunity (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. 2012. Am J Hum Genet 90, 986-1001; Burns, et al., LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia. 2012. J Allergy Clin Immunol 130, 1428-32; Alangari, et al., LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency. 2012. J Allergy Clin Immunol 130, 481-8 e2); depletion of LRBA results in significant cell growth inhibition, and sensitizes cells to apoptosis (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity 2012. Am J Hum Genet 90, 986-1001; Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97); LRBA is important for cell proliferation, apoptosis and autophagy (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. 2012. Am J Hum Genet 90, 986-1001; Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97); and deficiency of LRBA results in fewer cells positive for three CVID proteins: CD19, CD20 and B cell-activating factor receptor (BAFFR). This suggests that it may regulate these genes, and may cause immunodeficiency at least partially through these genes (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. 2012. Am J Hum Genet 90, 986-1001).
LRBA appears to regulate cytokines. LRBA-deficient patients have immunodeficiency, and defective cytokines are known to cause immunodeficiency (Leonard, Cytokines and immunodeficiency diseases. 2001. Nat Rev Immunol 1, 200-8). LRBA paralogue, FAN (Factor associated with neutral sphingomyelinase activation) is a TNF adaptor protein and is required for the TNF-induced expression of cytokines, such as IL-6 and CXCL-2 (Adam-Klages, et al., FAN, a novel WD-repeat protein, couples the p55 TNF-receptor to neutral sphingomyelinase. 1996. Cell 86, 937-47; Montfort, et al., FAN stimulates TNF(alpha)-induced gene expression, leukocyte recruitment, and humoral response. 2009. J Immunol 183, 5369-78). LYST is required for LPS induction of inflammatory cytokines (Cheng, et al., Novel functions of a regulator of lysosomal trafficking, LYST in TLR4 signal transduction. 2012. Immunology 137, 299-300).
These findings demonstrate that LRBA is essential for proper functioning of the immune system. However, the underlying cellular and molecular mechanisms are most unknown. LRBA may be an effective therapeutic target for inflammatory diseases. For example, RA is a severe, often destructive, chronic inflammatory disease of peripheral joints. Anti-TNF therapy has revolutionized the treatment of RA and other inflammatory diseases (Tracey, et al., Tumor necrosis factor antagonist mechanisms of action: a comprehensive review. 2008. Pharmacol Ther 117, 244-79). However, anti-TNF therapy is not effective in about one-third of RA patients and most of other inflammatory diseases, and patients may become refractory to the treatment. It may have severe side effects, and can also cause or increase heart failure (Ambrosi, & Lafforgue, [The cardiologist and immunosuppressive therapy]. 2012. Presse Med 41, 655-61; Nicola, et al., The risk of congestive heart failure in rheumatoid arthritis: a population-based study over 46 years. 2005. Arthritis Rheum 52, 412-20). New therapeutic strategies, based on a large number of cytokines, for these patients unresponsive to anti-TNF therapy have been explored, but the results are disappointing in most cases. The trials using IL-5 antagonists are disappointing (Commins, et al., Immunologic messenger molecules: cytokines, interferons, and chemokines. 2010. J Allergy Clin Immunol 125, S53-72). This may reflect that the cytokine cascade is highly redundant, and an inflammatory disease is often a complex disease, multiple cytokines may be involved in the inflammation (Holgate, S. T. Novel targets of therapy in asthma. Curr Opin Pulm Med 15, 63-71 (2009)). Inhibition of a single cytokine may not be sufficient, whereas blocking many cytokines with multiple agents would be impractical. Targeting a crucial gene that regulates multiple cytokines is desirable. LRBA may be one of such genes, as it may be a crucial regulator of multiple immune effectors including secreted cytokines through vesicle trafficking.
LRBA appears to regulate TLR4, as TLR4/LPS complex rapidly cycles between the cell membrane, the GC and endosomes (Latz, et al., Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction. 2002. J Biol Chem 277, 47834-43; Thieblemont & Wright, Transport of bacterial lipopolysaccharide to the golgi apparatus. 1999. J Exp Med 190, 523-34), and can activate signaling pathways at endolysosome (Cheng, et al., Novel functions of a regulator of lysosomal trafficking, LYST in TLR4 signal transduction. 2012. Immunology 137, 299-300), indicating vesicle-mediated localization and trafficking of TLR4 are required for its activation. Lyst deficient cells exhibit defective TLR signaling, specifically in TLR4 pathways (Cheng, et al., Novel functions of a regulator of lysosomal trafficking, LYST in TLR4 signal transduction. 2012. Immunology 137, 299-300). Recurrent bacterial infections in LRBA-deficient patients indicates impaired TLR4/LPS pathway. In summary, LRBA may interact with multiple important signal transduction pathways, including epidermal growth factor receptor (EGFR), Notch, PKA, Ras, E2F1, p53, MAPK, NF-κB LPS, cytokines and TLRs (Schreiber, et al., Genetic screen for modifiers of the rough eye phenotype resulting from overexpression of the Notch antagonist hairless in Drosophila. 2002. Genesis 33, 141-52; Shamloula, et al., rugose (rg), a Drosophila A kinase Anchor Protein, Is Required for Retinal Pattern Formation and Interacts Genetically With Multiple Signaling Pathways. 2002. Genetics 161, 693-710; de Souza, et al., SEL-2, the C. elegans neurobeachin/LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells. 2007. Development 134, 691-702; Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97). This concept is supported by information showing LRBA is highly conserved throughout the eukaryotic kingdom; LRBA interacts with multiple important signal transduction pathways, including those of epidermal growth factor receptor (EGFR), Notch, PKA, Ras, E2F1, p53 and NF-κB (Schreiber, et al., Genetic screen for modifiers of the rough eye phenotype resulting from overexpression of the Notch antagonist hairless in Drosophila. 2002. Genesis 33, 141-52; Shamloula, et al., rugose (rg), a Drosophila A kinase Anchor Protein, Is Required for Retinal Pattern Formation and Interacts Genetically With Multiple Signaling Pathways. 2002. Genetics 161, 693-710; de Souza, et al., SEL-2, the C. elegans neurobeachin/LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells. 2007. Development 134, 691-702; Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97); mutations of LRBA cause severe immunodeficiency and autoimmunity (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. 2012. Am J Hum Genet 90, 986-1001; Burns, et al., LRBA gene deletion in a patient presenting with autoimmunity without hypogammaglobulinemia. 2012. J Allergy Clin Immunol 130, 1428-32; Alangari, A. et al. LPS-responsive beige-like anchor (LRBA) gene mutation in a family with inflammatory bowel disease and combined immunodeficiency. 2012. J Allergy Clin Immunol 130, 481-8 e2); depletion of LRBA results in significant cell growth inhibition, and sensitizes cells to apoptosis (Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97; Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. 2012. Am J Hum Genet 90, 986-1001); LRBA is important for cell proliferation, apoptosis and autophagy (Wang, et al., Deregulated expression of LRBA facilitates cancer cell growth. 2004. Oncogene 23, 4089-97; Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. 2012. Am J Hum Genet 90, 986-1001) and deficiency of LRBA causes less cells positive for at least three CVID proteins: CD19, CD20 and BAFFR, suggesting that LRBA may regulate these genes (Lopez-Herrera, et al., Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity. 2012. Am J Hum Genet 90, 986-1001).